Cells culture and EGFP stable expression
Human embryonic kidney (HEK293) cells were from American Type Culture Collection (ATCC CRL-1573, Manassas, USA) and cultured in Dulbecco’s modified Eagle’s Medium (DMEM, Sigma) supplemented with fetal bovine serum (10%) and penicillin/streptomycin (Gibco, Life Technologies). Cells were maintained at 37oC and 5% CO2 in a humidified incubator. To stably express EGFP in HEK293, cells were seeded in 12-well plates with 1ml of DMEM. When cells reached 60–80% confluency, medium was replaced with Opti-MEM (Gibco, Life Technologies). Then cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. One μg of pEGFP-N1 was transfected with 2μL Lipofectamine 2000. Medium was replaced with fresh DMEM medium with serum 6hrs after transfection. 48hrs later, cells were treated with G418 (500 μg/mL, Sigma) for 15 days with medium changed every 3 days. Colonies were picked into 96 wells and expanded into 6-well plates before genomic DNA extraction, PCR amplification, and sequencing.